The cell cytosol is crowded with macromolecules such as proteins, nucleic acids, and membranes. The consequences of such crowding remain unclear. How is the rate of a typical enzymatic reaction, involving a freely diffusing enzyme and substrate, affected by the presence of macromolecules of different sizes, shapes, and concentrations? Here, we mimic the cytosolic crowding in vitro, using dextrans and Ficolls, for the first time in a variety of sizes ranging from 15 to 500 kDa, in a concentration range 0-30% w/w. Alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate (PNPP) was chosen as the model reaction. A pronounced decrease in the rate with increase in fractional volume occupancy of dextran is observed for larger dextrans (200 and 500 kDa) in contrast to smaller dextrans (15-70 kDa). Our results indicate that, at 20% w/w, smaller dextrans (15-70 kDa) reduce the initial rate moderately (1.4- to 2.4-fold slowing), while larger dextrans (>200 kDa) slow the reaction considerably (>5-fold). Ficolls (70 and 400 kDa) slow the reaction moderately (1.3- to 2.3-fold). The influence of smaller dextrans was accounted by a combination of increase in viscosity as sensed by PNPP and a minor offsetting increase in enzyme activity due to crowding. Larger dextrans apparently reduce the frequency of enzyme substrate encounter. The reduced influence of Ficolls is attributed to their compact and quasispherical shape, much unlike the dextrans.

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